Prevalence and distribution of subtypes of Blastocystis in Asiatic brush-tailed porcupines (Atherurus macrourus), bamboo rats (Rhizomys pruinosus), and masked palm civets (Paguma larvata) farmed in Hainan, China

Blastocystis sp. is an important gastrointestinal parasite with global distribution, prevalent in humans, farmed animals, and wildlife. Therefore, this study aimed to investigate the prevalence and genetic diversity of Blastocystis sp. in Asiatic brush-tailed porcupines (Atherurus macrourus), bamboo rats (Rhizomys pruinosus), and masked palm civets (Paguma larvata) in Hainan Province, China. A total of 900 fecal samples were collected from three farmed animal species including 257 porcupines, 360 rats, and 283 civets. Genomic DNA was extracted from each fecal sample and Blastocystis sp. was detected by PCR at the small subunit ribosomal RNA (SSU rRNA) gene. A phylogenetic tree was constructed using the maximum likelihood method. Blastocystis sp. was detected in 47 (5.2%) fecal samples: 12 (4.7%) Asiatic brush-tailed porcupines, 8 (2.2%) bamboo rats, and 27 (9.5%) masked palm civets. Three known Blastocystis sp. subtypes, including ST1, ST4, ST5, and one unnamed subtype (unST), were found in one, 19, 26, and one animal, respectively. Subtypes ST4 and unST were detected in porcupines, ST4 in rats, and ST1 and ST5 in civets. Our results suggest that the three farmed animal species reported in this study could serve as reservoirs for potentially zoonotic Blastocystis sp. subtypes and transmit this parasite to humans, other farmed animals, and wildlife.


Introduction
Blastocystis sp. is a ubiquitous gut parasite that causes intestinal infections in humans and almost all animal species [12,26].The pathogenicity of Blastocystis sp. is controversial, and Blastocystis sp.infection is generally asymptomatic.Nonetheless, symptoms such as diarrhea, abdominal pain, weight loss, and bloating may occur in some patients [2].Zoonotic, food and waterborne, and human-to-human transmission are the main routes through which Blastocystis sp. is transmitted [32].In most instances, infection occurs via the fecal-oral route by drinking or eating the Blastocystis sp.cysts in contaminated food or water [21].Identifying the source of Blastocystis sp.infections is key in preventing and controlling the spread of the parasite.However, there are limited data on the role of farmed animals in transmitting Blastocystis sp. to humans.
Asiatic brush-tailed porcupines (Atherurus macrourus), bamboo rats (Rhizomys pruinosus), and masked palm civets (Paguma larvata) have recently been assessed for the IUCN Red List of Threatened Species, and in China, they are commonly farmed for food in Hainan Province.However, the role of these animals for Blastocystis sp. is not well understood.Therefore, this study used PCR to investigate the prevalence and genetic diversity of Blastocystis sp. in Asiatic brush-tailed porcupines, bamboo rats, and masked palm civets in Hainan Province to assess their zoonotic potential.

Ethics statement
This study was approved by the Research Ethics Committee and the Animal Ethical Committee of Wenzhou Medical University.Only excreted feces from the study animals were analyzed.No animal was injured during the research.

Sample collection
A total of 900 fecal samples were collected from 257 Asiatic brush-tailed porcupines, 360 bamboo rats, and 283 masked palm civets farmed in Hainan Province between December 1, 2017, and March 31, 2021 (Table 1).Asiatic brush-tailed porcupine samples were obtained from 6 farms in Baisha, Baoting, Lingshui, Wuzhishan, Dingan, and Qiongzhong; those of bamboo rats were sampled from 3 farms in Danzhou, Dingan, and Qiongzhong; and those of masked palm civets were sampled from 5 farms in Baisha, Dingan, Haikou, Lingshui, and Wuzhishan (Fig. 1).To minimize duplicate sampling, only one fecal sample (~15 g) was collected in each animal cage (each with 1 to 3 animals).Approximately 30-50% of animals on each farm were sampled.The farms were selected based on the willingness of the owners to participate in the study.All farms had a relatively good standard of environmental sanitation where animals are provided with clean tap water for drinking, 1 to 3 animals were kept in individual cages with minimal contact with other species.The management of animals on all fourteen farms was similar.Fresh fecal samples were collected in labeled sterile plastic bags and transported to the Key Laboratory of Tropical Translational Medicine of the Ministry of Education (Hainan Medical University) in containers packed with ice packs within 24 h and stored at 4 °C for processing within 12 h.None of the animals showed diarrhea at the time of sample collection.

DNA extraction
Each fecal sample was sieved, and the filtrates were washed three times with distilled water and then centrifuged for 10 min at 1500 Âg to concentrate the parasites.Genomic DNA was extracted from 180-200 mg of clean fecal samples using a QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions.The extracted DNA was eluted in 200 lL of AE buffer and stored at À20 °C prior to PCR analysis.

PCR amplification
The bamboo rats were identified to the species level by amplification of a 421-bp region of the cytb gene from fecal DNA using PCR [33].To detect Blastocystis sp., an approximately 500 base pair fragment of the SSU rDNA gene was amplified by PCR as described by Santin et al. [29].TaKaRa Taq DNA Polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for all PCR amplifications.Negative controls were included in all PCR tests for quality control.The PCR products were subjected to 1.5% agarose gel electrophoresis and were visualized on a Gel Doc EZ UV-gel imaging system (Bio-Rad Inc., USA).The gel was stained with GelRed (Biotium Inc., Hayward, CA) to aid in visualization.

Nucleotide sequencing and analysis
The PCR products of interest were sequenced by Sangon Biotech Co., Ltd (Shanghai, China).Sequence accuracy was confirmed by two-directional sequencing and further sequencing of PCR products for some DNA samples where necessary.Sequences of each strand were edited and aligned using DNASTAR Lasergene EditSeq v7.1.0(http://www.dnastar.com/) and Clustal X v2.1 (http://www.clustal.org/)tools.Reference sequences were downloaded from the GenBank.

Phylogenetic analysis
Mega 7 was used to produce a phylogenetic tree for nucleotide sequences using the maximum likelihood method.One thousand bootstrap replicates were used to test the phylogenetic tree's reliability and the statistical support for the topology.Evolutionary distances were calculated using the Tamura-3 parameter model [16].

Nucleotide sequence accession numbers
The novel nucleotide sequence of Blastocystis ST5 obtained in this study was deposited in the GenBank database under accession number: OP132525.Meanwhile, all the other sequences which similarity of 100% with sequences available in GenBank obtained here were submitted to GenBank under accession number: OR593325 for ST1, OR593326 to OR593328 for ST4, OR593329 for unST, and OR593330 to OR593339 for ST5.

Statistical analyses
Data were analyzed using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA).The chi-square test at 95% confidence

Results
Infection rates of Blastocystis sp.
Blastocystis sp. was detected in 47 (5.2%) of the 900 fecal samples from 27 (9.5%)masked palm civets, 12 (4.7%)Asiatic brush-tailed porcupines, and 8 (2.2%) bamboo rats (Table 1).Notably, the differences in the prevalence rates of Blastocystis sp.among the three species and across the farms sampled were statistically significant (v 2 = 17.4,df = 2, p < 0.001).Positive fecal samples of Asiatic brush-tailed porcupines were from four of the six farms surveyed, with the highest infection rate (20.0%) in Wuzhishan, followed by Baoting, Lingshui, and Baisha (Table 1).Positive fecal samples of bamboo rats and masked palm civets were from one out of all the farms surveyed.The prevalence of Blastocystis sp. in bamboo rats in Danzhou farm was 7.0%, and 48.2% in masked palm civets in Lingshui farm (Table 1).

Sequencing of PCR amplicons
PCR and sequencing analysis of cytb amplicons showed that all the bamboo rats were Rhizomys pruinosus and all cytb gene sequences had 100% identity with the reference sequence KY754146.Meanwhile all the 47 PCR amplicons were sequenced to determine Blastocystis sp.subtypes.Nucleotide sequence analysis revealed the presence of three distinct previously reported subtypes (ST1, ST4, and ST5) and one unnamed subtype (unST); there were no mixed infections detected.Among them, ST5 was the most common subtype detected at 55.3% (26/47), followed by ST4 at 40.4% (19/47).The prevalence of ST1 and the unST were each at 2.1% (1/47).While ST4 was detected in both Asiatic brush-tailed porcupines and bamboo rats, ST1 and ST5 were detected in masked palm civets only, and the unST was detected in one Asiatic brush-tailed porcupine (Table 1).

Discussion
In the present study, Blastocystis sp. was detected in 5.2% (47/900) of animals, including 4.7% (12/257) Asiatic brushtailed porcupine, 2.2% (8/360) bamboo rats, and 9.5% (27/283) masked palm civets sampled (Table 1).No study has reported on Blastocystis sp. in Asiatic brush-tailed porcupines and masked palm civets.Also, only one study has reported Blastocystis sp.infection in bamboo rats [31].Unlike the low infection rate of 2.2% in bamboo rats sampled in this study, the single previous study on the same animal species from this area reported a higher prevalence rate of 4.58% [31].Analysis of previous studies on the prevalence of Blastocystis sp. in wild and farmed animal species such as rodents and carnivores indicate geographical variations in infection rates, ranging from 3.1-100% [1, 3-10, 11, 13-15, 17-20, 22-25, 27, 28, 31, 34-36].The differences in infection rates are possibly due to host variation in different regions.The prevalence of Blastocystis sp. in the two farmed rodents investigated in the present study was lower compared to other farmed rodents, such as in Myocastor coypus (14.3%) and in flying squirrels (30.4%) in China [9,34], and in guinea pigs (35.4%) from Ecuador [11].In addition to the animal species, the sample size, the health of the animals, the environment, geographical location, and the type of detection method may explain the variability in the prevalence rates.It is worth noting that only one study was done in 12 out of the 15 countries for rodents and seven out of eight countries for carnivores with extremely small sample sizes [1, 3-10, 11, 13-15, 17-20, 22-25, 27, 28, 31, 34-36].The presence of Blastocystis sp. in rodents and carnivores is a public health concern, requiring large-scale investigations to assess their role in transmitting this parasite to humans.
ST5 was the most prevalent Blastocystis sp.subtype in this study (55.3%).This subtype has previously been detected in multiple animal hosts, mostly pigs, with a 1.64% infection rate reported in human samples worldwide [26].The ST5 prevalence rate in farmed masked palm civets suggests zoonotic transmission of this parasite from these animals to humans, pigs, and other animals, or, conversely, the ST5 harbored in the masked palm civets may also come from pigs or humans.Almost 95.0% of Blastocystis sp.detected in bamboo rats and porcupines were ST4.The global Blastocystis sp.ST4 prevalence in humans is 5.9%, although in Europe it is responsible for 19.8% of the cases reported [26].This subtype has also been detected in rodents, birds, several deer species, and other wild mammals worldwide, indicating that it has a wide host range [27].Given the prevalence of Blastocystis sp.ST4 in rodents (>17 rodent species), the subtype has possibly adapted to infect this type of host.Since most rodent species are peridomestic, they are considered potential sources of human Blastocystis sp.infections, needing extensive investigation.Although ST1 was only detected in one sampled animal (2.1%), the subtype is an important public health parasite because it is the second most common Blastocystis sp.subtype infecting humans and has been detected in animals worldwide [12,26].The unST detected in the present study has 92.4% identity with ST14.Given that this unST has been detected in a coypu in Henan [9], it potentially infects multiple hosts, including coypu and Asiatic brush-tailed porcupine.

Conclusion
This is the first report on the prevalence and genetic diversity of Blastocystis sp. in Asiatic brush-tailed porcupines, bamboo rats, and masked palm civets in Hainan Province.Our results indicate circulation of three known Blastocystis sp.subtypes (ST1, ST4, and ST5) and an unnamed Blastocystis sp.subtype in the animals mentioned above.These findings could provide a basis for preventing and controlling Blastocystis sp.infections in humans and the farmed animals in this region.

Figure 1 .
Figure 1.A map depicting the sampling sites for collecting fecal samples from Asiatic brush-tailed porcupines, bamboo rats and masked palm civets in Hainan Province, China.

Figure 2 .
Figure 2. Sequence variation in the small subunit (SSU) rRNA gene among of Blastocystis sp.ST5.SSU rRNA sequences of ST5 identified in this study were aligned.MN472792 as the reference sequence.

Figure 3 .
Figure 3. Phylogenetic relationship of Blastocystis sp.STs based on SSU rRNA gene sequences.Clustal X and Mega 7 software were used to calculate genetic distance and build the phylogenetic tree.The phylogenetic tree was constructed using the maximum likelihood method, based on the evolutionary distances calculated by the Tamura-3 parameter model; all positions containing gaps and missing data were eliminated.Bootstrap values were obtained using 1,000 replicates.The blue and red fonts represent the known and novel sequences, respectively identified in this study.

Table 1 .
Prevalence and distribution of STs of Blastocystis in the three farmed animals in Hainan Province, China.